Ligand and genetic studies of the sulfate-binding protein and characterization of the calcium-binding site in the galactose-binding protein

Author

Jacobson, Bruce Lee

Date

1990

Advisor

Quiocho, Florante A.

Degree

Doctor of Philosophy

Abstract

The effect of pH on the binding affinities of the conjugate bases of four different tetrahedral oxyacids to the periplasmic sulfate-binding protein from Salmonella typhimurium has been determined. In light of the highly refined 2 A structure of the complex of the sulfate-binding protein with sulfate, and considering the protonation state and net charge of the various oxyacids, the pH dependence of chromate binding and the extremely low affinity of phosphate are attributable mainly to a lack of hydrogen bond acceptors in the binding site. These studies demonstrate that the binding site of the sulfate-binding protein is stringently designed to tightly bind tetrahedral, fully ionized, oxyacid dianions.
Based on the refined 2.0 A structure of the periplasmic sulfate-binding protein from Salmonella typhimurium, twelve site-directed mutants in the E. coli periplasmic sulfate-binding protein were designed to test specific hypotheses regarding protein-ligand complex stabilization and binding mechanism.
Mutants at position 42 (H42N, H42G, H42D) demonstrate that the fidelity of the H-bond array to His 42 is more important in stabilizing the protein-SO$\sb{4}\sp{2-}$ complex than the proposed positive charge. The structural and chemical differences between serine and cysteine were examined by the series of mutants S130G, S130A, S130C. Mutations in the interdomain salt-bridges formed in the closed conformation of the protein were shown to affect the sulfate on- and off-rates. The necessity for protein conformational change in sulfate binding and release was tested by introducing a disulfide between the two domains. These studies further illuminate binding protein specificity, complex stability, and flexibility.
The relative affinities for various metals which bind to the calcium-binding site of the E. coli periplasmic $\sc {D}$-galactose-binding protein in solution have been determined. In order of affinity the metals are: Ca$\sp{2+}$ $\approx$ Tb$\sp{3+}$ $\approx$ Pb$\sp{2+}$ $>$ Cd$\sp{2+}$ $>$ Sr$\sp{2+}$ $>$ Mg$\sp{2+}$ $\gg$ Mn$\sp{2+}$ $>$ Ba$\sp{2+}$. The results of these solution studies support the hypothesis that for a given metal-binding loop, the ligands provided by the protein, and the cation hydration energy, size, and charge are major factors contributing to binding affinity.

Keyword

Molecular biology; Biophysics; Biology

Citation

Jacobson, Bruce Lee. "Ligand and genetic studies of the sulfate-binding protein and characterization of the calcium-binding site in the galactose-binding protein." (1990) PhD diss., Rice University. http://hdl.handle.net/1911/16352.